Summary: A.) Further Studies with isolated absolute infrared (IR) spectra of Bacteriorhodopsin (BR) Photocycle intermediates. In the previous report, we described the isolation for the first time of absolute BR photocycle intermediates spectra, which were then used to trace the aminoacid residue binding sites for protons as they are electrogenically pumped across the membrane. Since then we have focused our attention on following conformational changes of the BR protein during the pumping process. This is possible because there are three specific regions in the IR spectrum where environmental influences on the characteristic frequencies of the peptide bond are seen. These are known as Amide I, Amide II, and Amide III. Our goal was to quantify the magnitudes of these changes at specific frequencies within the broad envelopes of the three regions. Our results from all three regions were consistent in finding that the magnitudes of conformational changes reach a maximum in the formation of the N intermediate, and are then reversed in the return to ground state through the N to O to BR transitions. These studies were completed and submitted for publication. B.) Development of instrumentation and procedures for comparing visible and IR kinetics of the BR photocycle in membrane protein crystals to that of in situ in tiny membrane fragments. Crystals of functional proteins are widely perceived and used as models for how proteins act in vivo. This is a particularly tenuous assumption for membrane proteins because they are most often prepared using detergents. We think it is essential to establish how such crystals mimic the activities of the in situ protein. This is a joint project with NIST. For the past few years, we have been developing instrumentation and procedures using visible spectroscopy at NIH. In the earlier part of the year we managed to solve most of the problems (mentioned in our 2011 annual report) with our image intensifier and charge-coupled device (CCD)camera/spectrometer. In order to not harm the CCD, we must work at a low level of monitoring light, which lowers the signal/noise(S/N) ratio. To compensate for this, we have been able to modify both the hardware interfaces and software to increase our visible data collection rate from 0.33 to 10 Hz. This plus longer collection times improves the S/N ratios to workable levels. At NIST, we have been simultaneously developing the IR instrumentation and procedures using a powerful IR microscope, which we adapted to accept visible light passed through the same IR-monitored sample and sends it to the CCD/spectrometer, using fiber optics. Several months ago, we moved the visible half of the system to NIST where the two units are being integrated for the combined vis/IR approach. A NIST crystallographer is a collaborator, and we are exploring procedures for forming and isolating a large number of membrane BR crystals. This is important for two reasons. 1. The crystals are formed in a highly viscous medium, and are easily damaged in trying to extract them. 2. Crystals are damaged by Laue X-ray irradiation, and they must be changed frequently during the time-resolved data collection period. Phillip Anfinrud of NIDDK has agreed to collaborate with us if we could supply him with enough of the isolated crystals. Petra Fromme of Arizona State University is interested in a possible collaboration using femtosecond nanocrystallography and X-ray lasers, when we get to that point. C.) Studies on amyloidosis of amyloid beta (abeta) protein in Alzheimer's disease (AD) The polymerization of monomers of amyloid beta through oligomers, fibrils, and plaques is recognized in the pathology of AD. Like many other polymerization phenomena, the process shows a lag phase followed by the formation of a nucleus leading to a logarithmic growth of the polymer which ends in a plateau. Originally it was thought that the pathologyy resulted from the large fibrils and plaques, but it is currently believed that the culprit is a small soluble oligomer. A likely candidate is the nucleus. It is also known that the predominant protein conformational change in the early steps is from random coil to alpha-helix. The fibers and plaques are mainly beta-sheet. Time-resolved IR measurements provide a means recording conformational changes at all stages of the polymerization. SVD (developed in my laboratory) should then allow us to obtain a time profile of these changes. We would like to identify the protein conformation and shape of the oligomer (nucleus) which begins the processes leading to the loss of axon function in AD. During the past few months at NIST, we have established that there are sufficient IR signal to noise (S/N) ratios in the three amide regions, and system stability to track the kinetics of conformational changes. The ultimate goal to obtain an isolated IR spectrum for each stage in the polymerization requires our defining a precise kinetic model, expressed in a system of differential equations. To do this we need additional information on the size and shape of the successive intermediates. To this end, I have assembled a group of highly talented researchers at NIH who have agreed to collaborate with us. Patrick Brown (NIBIB) who will use dynamic light scattering (DLS) and circular dichroism (CD). Albert Jin (NIBIB) who will use atomic force microscopy (AFM). Richard Leapman (SD, NIBIB) who will use electron microscopy (EM) Paul D. Smith (NIBIB) who will manage all required optics and also perform normal light scattering (LS). Youngchan Kim (Computational Metaphysics System Lab, NRL) who has had much experience in deducing kinetic models for polymerization processes. Next steps: We will make a standardized bulk abeta preparation and distribute it in small tubes so that upon dilution in our peristaltically pumped closed circuit at NIST, the concentration will be 1 mg/ml. The same tubes will be independently monitored at NIH using the exact conditions as the IR monitored incubations at NIST. During the incubations at NIH, small (10 microL) aliquots will be taken for immediate measurements by DLS, CD, AFM, and EM. The linkage to align the kinetics performed at NIH with those at NIST will be LS. This will allow the matching of time profiles for the intermediates in the two sets of data. Some important background: With many collaborators, my laboratory previously demonstrated that potent antibacterial substances found in the skin of Xenopus laevis were small oligomers of amphiphilic alpha-helical peptides in the size range of abeta. We showed that these oligomers formed channels in the membranes of bacteria, tumor cells, and cytochrome oxidase liposomes leading to depolarization and the loss of electrochemical potentials. Several reports on early stages of amyloidosis point to depolarization of membranes. We believe that the abeta oligomers with high alpha-helical content may disrupt brain function by a similar mechanism. We currently see three distinct phases in the investigation: 1. Identification of the oligomers with high alpha-helical content. 2. Repeat the experiments of phase 1, but in the presence of liposomes to see if the time profiles of oligomer sizes and protein conformations are the same. 3. Repeat experiments of phase 2, but preload the liposomes with 50 mM NaCl, and suspend them in a medium with 50 mM KCl to simulate a membrane potential comparable to that of an energized axon after hydrolysis of ATP to form the gradient. Have K+/Na+ selective electrodes present to measure the leakage rate of the two cations into and out of the liposomes. The object is to see if the exchange rate rapidly increases when the alpha-helical oligomers are formed.